Back to EveryPatent.com
| United States Patent |
6,046,034
|
|
Waschutza
, et al.
|
April 4, 2000
|
Thermostable variants of human interferon-.gamma.(IFN-.gamma.)
Abstract
The invention provides new variants of recombinant human interferon-.gamma.
(rhIFN-.gamma.), vectors and host cells for their production, and
therapeutic methods employing them. The variants are characterized by the
substitution of one or more pairs of amino acids selected from Glu.sup.8
-Ser.sup.70, Ala.sup.18 -His.sup.112, Lys.sup.81 Leu.sup.121, and
Gln.sup.49 -Leu.sup.96 by pairs of Cys residues, and optionally by the
deletion of from one to ten amino acid residues from C-terminus of the
native IFN-.gamma. sequence. The variants of the invention exhibit greater
thermal stability and no loss of biological activity as compared to
native-sequence rhIFN-.gamma..
| Inventors:
|
Waschutza; Gero (Meinersen, DE);
Li; Volkhart (Cologne, DE);
Otto; Bernd (Hannover, DE)
|
| Assignee:
|
Fraunhofer-Gesellschaft zur Forderung der Angewandten Forschung E. V. (Munich, DE)
|
| Appl. No.:
|
029819 |
| Filed:
|
May 11, 1998 |
| PCT Filed:
|
August 15, 1996
|
| PCT NO:
|
PCT/DE96/01556
|
| 371 Date:
|
May 11, 1998
|
| 102(e) Date:
|
May 11, 1998
|
| PCT PUB.NO.:
|
WO97/24376 |
| PCT PUB. Date:
|
July 10, 1997 |
Foreign Application Priority Data
| Sep 18, 1995[DE] | 195 35 863 |
| Current U.S. Class: |
435/69.51; 424/85.5; 435/252.3; 435/252.33; 435/320.1; 435/325; 530/351; 536/23.52 |
| Intern'l Class: |
C12P 021/04; C07K 021/04; C07K 001/00; A61K 038/21 |
| Field of Search: |
536/23.52
530/351
424/85.5
435/69.51,325,252.3,252.33,320.1
|
References Cited
| Foreign Patent Documents |
| 0 170 917 A1 | Feb., 1986 | EP.
| |
| 0 306 870 A2 | Mar., 1989 | EP.
| |
| 92/08737 | May., 1992 | WO.
| |
Other References
Modelling of Interhelical Contacts in Interferons-.beta., -.gamma., and
Dric Interleukin-5, Biochemical and Biophysical Research Communications,
vol. 201, No. 3, Jun. 30, 1994, pp. 1401-1405.
Expression of human immune interferon cDNA in E.coli and monkey cells,
Nature, vol. 295, Feb. 11, 1982, pp. 503-508.
Molecular cloning of human immune interferon cDNA and its expression in
eukaryotic cells, Nucleic Acids Research, vol. 10, No. 8, 1982, pp.
2487-2501.
Three-Dimensional Structure of Recombinant Human Interferon-.gamma.,
Science, vol. 252, May 3, 1991, pp. 698-702.
|
Primary Examiner: Fitzgerald; David L.
Attorney, Agent or Firm: Barnes & Thornburg
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national application of international
application Ser. No. PCT/DE96/01556, filed Aug. 15, 1996, which claims
priority to German serial No.195 35 853.8, filed Sep. 18, 1995.
Claims
We claim:
1. A substituted interferon-gamma (IFN-.gamma.) protein comprising:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2
wherein
Xaa at position 8 is Glu or Cys,
Xaa at position 70 is Ser or Cys,
Xaa at position 18 is Ala or Cys,
Xaa at position 112 is His or Cys,
Xaa at position 81 is Lys or Cys,
Xaa at position 121 is Leu or Cys,
Xaa at position 49 is Gln or Cys, and
Xaa at position 96 is Leu or Cys,
wherein Cys residues are present at both positions of at least one pair
selected from the group consisting of positions 8 and 70, 18 and 112, 81
and 121, and 49 and 96;
(b) a polypeptide which differs from a polypeptide according to (a) by the
deletion of 1 to 10 amino acids from the C-terminus; or
(c) a homodimer of a polypeptide according to (a) or (b).
2. A DNA molecule comprising a sequence that codes for a polypeptide
according to claim 1.
3. A recombinant vector, comprising a DNA sequence that codes for a
polypeptide according to claim 1.
4. The recombinant vector of claim 3, wherein the vector is a plasmid.
5. The recombinant vector of claim 4, wherein the plasmid is pKK233-2.
6. The recombinant vector of claim 3, wherein the vector is a
bacteriophage.
7. The recombinant vector of claim 6, wherein the bacteriophage is M13mp18.
8. A recombinant micro-organism, transformed with a vector according to one
of claims 3 to 7.
9. The recombinant micro-organism of claim 8, wherein the micro-organism is
a bacterium.
10. The recombinant micro-organism of claim 9, wherein the bacterium is
Escherichia coli K12 strain JM105.
11. A method of producing the recombinant bacteriophage of claim 6 or 7,
said method comprising the steps of digesting plasmid pKK233-2 containing
an IFN-.gamma.-encoding insert with a restriction endonuclease, inserting
the resulting DNA section coding for IFN-.gamma. into the DNA of
bacteriophage M13mp18, and modifying the IFN-.gamma. coding sequence to
encode said substituted IFN-.gamma. polypeptide.
12. The method of claim 11, wherein the plasmid is digested with the
restriction endonucleases EcoRl and HindIII.
13. A method of producing a recombinant bacterium comprising the step of
transfecting a bacterial cell with the plasmid of claim 4 or 5.
14. A method of producing the polypeptide of claim 1, said method
comprising the steps of cultivating a bacterium, transformed with a vector
according to one of claims 3 to 7, breaking open said transformed
bacterium and isolating the polypeptide.
15. The method of claim 14, wherein the bacteria cells are disrupted
mechanically.
16. The method of claim 14 wherein the isolation of the polypeptide
comprises the steps of washing the bacterial cell vesicles which contain
the polypeptide, solubilizing the polypeptide with guanidinium
hydrochloride, diluting the resulting solution, and renaturing the
polypeptide.
17. The method of claim 16, wherein the polypeptide is further isolated by
column or batch affinity chromatography followed by gel filtration.
18. The method of claim 17, wherein batch affinity chromatography is
conducted by bringing an affinity matrix into uniform contact with a
solution containing the polypeptide, washing the matrix, and eluting the
polypeptide from the matrix in batch format.
19. The method of claim 18, wherein the affinity chromatography matrix is
SP-SEPHAROSE.TM. or AFFI-GEL BLUE.TM..
20. A method of treating a patient having at least one of a kidney rumor,
chronic granulomatosis and neurodermatitis, said method comprising the
step of administering to said patient an effective amount of a composition
comprising a substituted IFN-.gamma. protein according to claim 1.
Description
The invention relates to variants of the human recombinant
interferon-.gamma. with increased thermal stability.
Interferon-.gamma. is suitable as an antiviral, antiproliferative,
immunomodulatory human therapeutic agent, particularly for the treatment
of kidney tumours and chronic granulomatosis.
Like the interleukins, interferons belong to the class of the cytokines and
are listed in various classes: interferon-.alpha., interferon-.beta.,
interferon-.gamma., interferon-.omega. and interferon-.tau..
Interferon-.gamma. is a glycoprotein, whose amino acid sequence has been
known since 1982 (Nucl. Acid Res. 10, pages 2487 and following (1982)). In
the mature condition the interferon-.gamma. has 143 amino acids and a
molecular weight of 63 to 73 kilodaltons. The amino acid sequence of
interferon-.gamma. is shown in FIG. 1. It will be noticed that the
sequence contains no cystein.
The tertiary and quaternary structure of the non-glycosylised protein was
clarified in 1991 (Science 252, page 698 and following (1991)). According
to this, interferon-.gamma. exists as a homodimer, the monomers being
orientated in contrary directions in such a way that the C-terminal end of
one monomer is located in the vicinity of the N-terminal end of the other
monomer. Each of these monomers in all has six .alpha.-helices.
Interferon-.gamma. is also called immunointerferon, as it has non-specific
antiviral, antiproliferative and in particular immunomodulatory effects.
Its production in T-helper-lymphocytes is stimulated by mitogens and
antigens. The effect of the expressed interferon-.gamma. has not yet been
precisely clarified, but is subject to intensive research. In particular,
interferon-.gamma. leads to the activation of macrophages and to the
synthesis of histocompatability antigens of the class 2. In vitro, the
activity of interferon-.gamma. is normally determined as a reduction in
the virus-induced cytopathic effect, which arises from treatment with
interferon-.gamma.. Due to its antigen-non-specific antiviral,
antiproliferative and immunomodulatory activity it is suitable as a human
therapeutic agent, for example of kidney tumours and chronic
granulomatosis. Clinical studies of various tumours are being carried out
at present. Great expectations are set also on the interferon-.gamma.
therapy of neurodermitis. Furthermore, interferon-.gamma. is used in the
research field also as a fine chemical for example for the stimulation of
cell cultures or for determining interferon levels.
Since 1982 it has been possible to express variants of the human
interferon-.gamma. in bacteria such as escherichia coli (Nature 295, pages
503 and following (1982)). The monomer of these recombinant variants of
the human interferon-.gamma. has, in addition to the 143 amino acids of
the native human monomer, an additional N-terminal methionin as an
additional amino acid. This recombinant human interferon-.gamma.
(rhu-IFN-.gamma.) is not glycosylised. It can be obtained in large
quantities and was therefore extremely accurately characterised.
The recombinant human interferon-.gamma. also exists as a dimer and has
normal biological activity. It is acid-sensitive and temperature-sensitive
with a melting point of 52.degree. C.
Of all the interferons, the thermal stability of human interferon-.gamma.
is the lowest. This low thermal stability of interferon-.gamma. renders it
difficult to use as a human therapeutic agent.
Tests were carried out at an early stage in order to generate variants with
improved properties of the recombinant human interferon-.gamma. with the
aid of molecular-biological methods. In EP 0 306870 A2, variants of
recombinant human interferon-.gamma. were presented, whose activity was
significantly increased by splitting off the C-terminal 7-11 amino acids.
In addition, WO 92-08737 indicates a variant of recombinant human
interferon-.gamma. (Interferon-.gamma. C-10L) which has an increased
biological activity. However, in none of these variants of recombinant
interferon-.gamma. was the thermal stability significantly increased.
Therefore the low thermal stability also renders difficult the use of
these proteins as a human therapeutic agent.
The object of the present invention is to make available variants of the
recombinant human interferon-.gamma. whose thermal stability is increased,
the biological activity being similar, preferably equal to or even better
than that of the unaltered recombinant human interferon-.gamma.. It is a
further object of this invention to indicate gene sequences, vectors with
gene sequences and micro-organisms with gene sequences which can be used
to manufacture the expression system and the variant according to the
invention of interferon-.gamma..
This object is achieved in that there are indicated DNA sequences and
recombinant vectors with such DNA sequences, for example plasmids and
bacteriophages, and micro-organisms which contain such DNA sequences,
which code for polypeptides, which are characterised in comparison to
recombinant human interferon-.gamma. by increased thermal stability.
Furthermore, methods are indicated by means of which such DNA sequences,
recombinant vectors and micro-organisms can be produced. With the aid of
the oligonucleotides according to the invention and corresponding
restriction endonucleases, on the basis of a plasmid known per se, which
contains the gene for recombinant human interferon-.gamma., recombinant
vectors such as plasmids or bacteriophages and micro-organisms can be
produced, which contain a DNA sequence which codes for the polypeptides
according to the invention.
There is further indicated a manufacturing method for the proteins
according to the invention, by means of which, with the aid of the
micro-organisms according to the invention the proteins according to the
invention can be obtained, concentrated and cleaned up, and uses of the
proteins according to the invention as a human therapeutic agent or/and
fine chemical.
The polypeptides according to the invention consist of a sequence of 144
amino acids, made up of the sequence of the 143 amino acids of the
recombinant human interferon-.gamma. and an additional N-terminal
methionin. Otherwise the polypeptide according to the invention differs
from the monomer of the recombinant human interferon-.gamma. in that at
least one pair of amino acids from four predetermined amino acid pairs is
exchanged for cystein. These four amino acid pairs are Glu8 and Ser70,
Ala18 and His112, Lys81 and Leu121 and Gln49 and Leu96. The complete amino
acid sequence is given for the recombinant human interferon-.gamma. in
FIG. 1 and for the polypeptide according to the invention in FIG. 2. In
this case Xaa means Glu8, Xab Ser70, Xba Ala18, Xbb His112, Xca Lys81, Xcb
Leu121, Xda Gln49 and Xdb Leu 96, insofar as these amino acids do not
stand for cystein in the polypeptide according to the invention.
It became surprisingly apparent that the proteins according to the
invention have higher thermal stability in comparison to recombinant human
interferon-.gamma..
The proteins according to the invention are probably, like recombinant
human interferon-.gamma., homodimers, whose monomers are so oriented that
the N-terminal end of one monomer is located in the vicinity of the
C-terminal end of the other monomer. Possibly the exchange in pairs of the
amino acids leads to additional intermonomeric and intramonomeric
disulphide bridges. Thus the exchange of Ala18 and His112 and the exchange
of Lys80 and Leu120 probably leads to intermonomeric disulphide bridges,
and the exchange of Glu8 and Ser70 to an intramonomeric disulphide bridge,
while the exchange of Glu49 and Leu96 does not lead to any additional
covalent bond. Clearly the introduction of cystein pairs into the amino
acid sequence of the polypeptide monomer cannot lead to a direct
conclusion regarding the formation of disulphide bridges.
Due to the improved properties of the proteins and polypeptides according
to claims 1, 2 and 3 compared to the previous recombinant human
interferon-.gamma., these proteins are particularly suitable for various
forms of application as a human therapeutic agent. They are usable with
advantage particularly in those areas in which previously recombinant
human interferon-.gamma. was used as a medicinal drug, such for example as
kidney tumours or chronic granulomatosis. It is further to be expected
that the proteins according to the invention can be used in all future
indications in which previous recombinant human interferon-.gamma. will be
used as a medicinal drug.
The variants according to the invention of the recombinant human
interferon-.gamma. are also suitable for use as fine chemical for example
for in vitro tests, in order to determine interferon levels and for the
stimulation of cell cultures. Due to their higher thermal stability and
thus their improved handling, and due to their antiviral,
antiproliferative and immunomodulatory biological activity, they can
replace previous recombinant human interferon-.gamma. in all areas where
the latter is used.
Further advantageous embodiments are illustrated in the dependent claims.
A shortening of the C-terminal end of the polypeptide monomer by one to ten
amino acids leads to an improvement in the biological activity. In this
way both the thermal stability of the variants according to the invention
of the recombinant human interferon-.gamma. and also its biological
activity are improved in comparison to recombinant human
interferon-.gamma..
The production of the new recombinant vectors and micro-organisms will be
described in the following.
In order to produce a recombinant micro-organism which contains a gene
sequence which codes for one of the polypeptides according to the
invention, the sequence of the human recombinant interferon-.gamma. is cut
with the aid of restriction endonucleases from a vector which contains
this sequence, and cloned into the DNA of a phage. The phage DNA is then
mutated with the aid of appropriate oligonucleotides, so that it codes for
one of the polypeptides according to the invention. The genetically
altered phage is then multiplied and the altered sequence for human
recombinant interferon-.gamma. is cut out of the DNA of the phage with the
aid of the same restriction endonucleases, and cloned back into the
plasmid. With the aid of this plasmid, a micro-organism is now
transfected, which can then express the new gene coding for the
polypeptide according to the invention.
This method will now be illustrated by the example of a transfected
bacterium escherichia coli K12 strain JM 105.
The known plasmid pKK-233-2/IFN-.gamma. was used as an outset material.
This plasmid was digested with the restriction endonucleases EcoRI and
HindIII. This plasmid has between the two intersections for EcoRI and
HindIII the gene for human recombinant interferon-.gamma. and a strong
IPTG-inducable trc-promoter. This DNA section with a length of 751 base
pairs was then likewise cloned with EcoRI and HindIII into the likewise
known bacteriophage M13mp18. Then the DNA of the bacteriophage was mutated
with methods known per se and appropriate oligonucleotides in such a way
that the gene for interferon-.gamma. codes for one of the variants
according to the invention. This new bacteriophage M13mp18/IFN-.gamma. was
multiplied in the bacterium escherichia coli K12 strain TG1. The now
altered sequence section, which contained the gene for the altered
rhu-IFN-.gamma. was cut out from the c-DNA of the multiplied phage with
the aid of the same restriction endonucleases EcoRI and HindIII, and
cloned back into the plasmid pKK233-2. This now likewise altered plasmid
pKK233-2/IFN-.gamma. was now used to transfect the bacterium escherichia
coli K12 strain JM105.
The oligonucleotides used have a length of 29 base pairs and a sequence
which is complementary to the single-strand DNA sequence of the
corresponding bacteriophage DNA which contains roughly centrally the base
pairs to be mutated. Both ACA and GCA were used as a complementary codon
for the amino acid cystein to be introduced. These oligonucleotide primers
were produced with a commercially available DNA synthesiser. No difference
was found in the mutation rate between the two codons for cystein.
In order to produce the proteins according to the invention, the
transformed recombinant micro-organism is cultivated and then the protein
according to the invention is separated, cleaned up and concentrated from
the culture.
The production method for the proteins according to the invention will now
be illustrated by way of example with reference to the abovementioned
transfected escherichia coli K13 strain JM105.
The transfected bacteria contain the gene sequence for the altered
interferon-.gamma. and a trc promotor inducible by IPTG. The bacteria are
taken in cultures and expression of the mutated interferon-.gamma. is
induced by addition of IPTG. The expressed altered interferon-.gamma. is
stored by the bacteria cells in so-called "inclusion bodies". In order to
clean the expressed altered interferon-.gamma., the bacteria cells after
successful expression are opened and the "inclusion bodies" are freed of
soluble bacterial proteins by multiple washing. The opening is undertaken
preferably mechanically, for example by ultrasound. The protein according
to the invention is brought into solution by a denaturing step with
guanidine hydrochloride and separated. Then the protein according to the
invention is renatured by dilution in a phosphate buffer, and folded into
its biologically active form. The interferon-.gamma., in this way pure to
more than 90%, is obtained at a rate of up to 30% of the total protein
content of the E.coli culture. It can then be concentrated and further
purified by an affinity chromatography on a column or in a batch-like
process and after a further filtration step, e.g. an HPLC-gel filtration,
achieves a purity of more than 95%.
A batch-like process in the sense of this invention, also known as a batch
method, is understood to mean the following: the chromatographic material
is regularly stirred into a solution of the protein according to the
invention in such a way that the altered interferon-.gamma. is uniformly
distributed and bonds to the chromatographic material and the
interferon-.gamma. protein concentration comes to no more than about 2
mg/ml packed chromatographic material. The chromatographic material laden
with interferon-.gamma. is washed in the batch with phosphate buffer and
the altered interferon-.gamma. is then eluated in the batch with a saline
solution in the phosphate buffer. In this case for example, SP-sepharose
or Affi-gel-blue can be used as chromatographic material.
The figures show:
FIG. 1: the amino acid sequence of the human recombinant human
interferon-.gamma. (SEQ ID NO: 1);
FIG. 2: the amino acid sequence of the variant of the recombinant human
interferon-.gamma. according to claim 1 (SEQ ID NO: 2);
FIG. 3: the amino acid sequence of the variants of the recombinant human
interferon-.gamma. according to claim 2; and
FIG. 4: the base sequence of the oligonucleotides, which were used to
produce the recombinant DNA which codes for the proteins according to the
invention.
Some embodiments of the invention, given by way of example, will be
explained in the following.
EXAMPLE 1
The amino acids Glu8 and Ser70 were exchanged according to the following
method and a correspondingly altered interferon-.gamma. was produced.
The interferon-.gamma. gene of the plasmid pKK-233-2/IFN-.gamma. was
recloned in the phages M13mp18. For this purpose a plasmid preparation was
digested with the restriction endonucleases EcoRI and HindIII. By means of
this digestion, the vector was split into two fragments with the lengths
751 base pairs and 4305 base pairs. Both fragments were separated from one
another via a 1% agarose gel, and the corresponding bands, of a size of
751 base pairs, were cut out from the gel and isolated. This fragment
contained, in addition to the complete interferon-.gamma.-gene, also the
trc-promoter and further vector ingredients at the 5' end.
A 150 ml culture of escherichia coli TG1 was infected with the phage
M13mp18. After incubation for 5 hours, the cells were centrifuged off,
resuspended once and again pelletised. The double-strand circular phage
DNA was cleaned up by the Quiagen.TM. plasmid isolation kit (Diagen
Company) from the cells. The RF-DNA of the phage M13mp18 was likewise
digested with the restriction enzymes EcoRI and HindIII and cleaned via a
1% agarose gel. Then the isolated EcoRI/HindIII insert, 751 bp long, was
cloned from the vector pKK-233-2/IFN-.gamma. in the phages M13mp18.
In order to produce expression vectors for the new variant of the
interferon-.gamma., mutations of the DNA sequence were carried out at
specific points. The method is based on hybridisation of an
oligonucleotide with the desired target sequence on single-strand DNA
(ssDNA) of the phage M13 to be altered. In order to produce the two spot
mutations of Glu8 and Ser70 to cystein, the following oligonucleotides
were used:
for the mutation of Glu8 to Cys
- TAAGGTTTTC TGCACATTTT ACATATGGG 29(SEQ ID NO:4)
- or
- TAAGGTTTTC TGCGCATTTT ACATATGGG 29(SEQ ID NO:5)
- and for mutation of Ser70 to Cys
- TGATGGTCTC CACACACTTT TGGATGCTC 29(SEQ ID NO:6)
- oder
- TGATGGTCTC CACGCACTTT TGGATGCTC 29(SEQ ID NO:7)
The oligonucleotides were produced with the aid of a commercially available
synthesiser. The two codons for cystein are TGT and TGC. The corresponding
complementary base sequence in the nucleotide strand is GCA or ACA. Both
codons were used (emphasised). As a length for oligonucleotide primers,
that of 29 base pairs was selected. All the nucleotides had to be
phosphorylised at their 5' end with T4-polynucleotidekinase for the
mutagenesis.
Independently of the sequence of the oligonucleotides, hybridisation was
carried out at 80.degree. C. in a water bath. After execution of the in
vitro mutagenesis, the transformation of the double-strand RF form of the
phage DNA was carried analogously to a normal plasmid with the CaCl.sub.2
method. Cells of E.coli were added to the recombinant phage-RF-DNA, IPTG
and X-Gal and plated out. After overnight incubation at 37.degree. C.
fourteen phage clones were respectively picked from each mutagenesis
reaction and again incubated together with E.coli for five hours at
37.degree. C.
The sequence monitoring of the mutated cDNA of the bacteriophages, which is
intended to code for the new variant of the recombinant
interferon-.gamma., was effected by didesoxy-sequencing, with the
T7-sequencing kit of Pharmacia. Thus the desired mutations were confirmed.
Following the mutagenesis of the phage-DNA, the sequence 751 bp long, which
coded for the altered rhu-IFN-.gamma., was cut out from the phage DNA with
the aid of the same restriction endonucleases, EcoRI and HindIII and
cloned back into the phages pKK233-2. The bacterium escherichia coli K12
strain JM105 was then transfected with this plasmid.
Expression of the altered interferon-.gamma. was then carried out according
to the methods described above. The biological, antiviral activity of the
altered interferon-.gamma. was determined by the reduction in the
virus-induced cytopathic effect which resulted after treatment with the
altered interferon-.gamma.. Human lung fibroblasts A549 were used as a
cell strain, and as a virus the encephalomyocarditis virus.
Analysis of the altered interferon-.gamma. obtained showed that its
biological activity was unaltered in comparison to recombinant human
interferon-.gamma.. In contrast to recombinant human interferon-.gamma.
with a melting temperature of 52.5.degree. C., the melting temperature of
the new variant was considerably increased at 68.5.degree. C. With an
identical biological activity, there consequently resulted for the new
variant of interferon-.gamma. a considerably improved thermal stability in
comparison to unaltered interferon-.gamma..
EXAMPLE 2
A variant of the recombinant human interferon-.gamma. was produced in which
the two amino acids Ala18 and His112 were exchanged for cystein.
Production of the corresponding DNA, phage DNA, plasmid DNA and the
corresponding expression organisms was carried out as in Example 1. The
following oligonucleotides were used for hybridisation of the ssDNA:
for the mutation of Alal8 to Cys
- CATCTGAATG ACCGCAATTA AAATATTTC 29(SEQ ID NO:8)
- or
- CA TCTGAATG ACCACAATTA AAATATTTC 29(SEQ ID NO:9)
- and for mutation of His112 to Cys
- ACTTGGATGA GTTCGCATAT TGCTTTGCG 29(SEQ ID NO:10)
- or
- ACTTGGATGA GTTCACATAT TGCTTTGCG 29(SEQ ID NO:11)
In this variant of recombinant human interferon-.gamma. also there was an
increased thermal stability with a melting point of the interferon-.gamma.
of 78.6.degree. C. with simultaneous indication of antiviral activity of
the new variant.
EXAMPLE 3
A variant of the recombinant human interferon-.gamma. was produced in which
the two amino acids Lys81 and Leu121 were exchanged for cystein.
Production of the corresponding DNA, plasmid-DNA, phage-DNA and of the
corresponding expression micro-organism was effected as described in
Example 1. The following oligonucleotides were used to produce the
variant:
for the mutation of Lys81 to Cys
- TGCTATTG-KA AAAACAGACA TTCATGTCT 29(SEQ ID NO:12)
- or
- TGCTATTGAA AAAGCAGACA TTCATGTCT 29(SEQ ID NO:13)
- and for mutation of Leu121 to Cys
- TAGCTGCTGG CGAACATTCA GCCATCACT 29(SEQ ID NO:14)
- or
- TA GCTGCTGG CGAGCATTCA GCCATCACT 29(SEQ ID NO:15)
- for the mutation of Gln49 to Cys
- AAAAGGAGAC A.ATGCAGCTC TGCATTATT 29(SEQ ID NO:16)
- or
- AAAAGGAGAC AATACAGCTC TGCATTATT 29(SEQ ID NO:17)
This variant of the recombinant human interferon-.gamma. revealed a
biological antiviral activity similar to that of the unaltered recombinant
interferon-.gamma. with improved thermal stability.
EXAMPLE 4
A variant of the recombinant interferon-.gamma. was produced in which the
two amino acids Gln49 and Leu96 were exchanged for cystein. Production of
the corresponding DNA, plasmid-DNA, phage-DNA and of the corresponding
expression micro-organism was carried out likewise as described in Example
1. The following oligonucleotides were used to produce this variant of
interferon-.gamma.:
for the mutation of Gln49 to Cys
- AAAAGGAGAC AATGCAGCTC TGCATTATT 29(SEQ ID NO:16)
- or
- AAAAGGAGAC AATACAGCTC TGCATTATT 29(SEQ ID NO:17)
- and for mutation of Leu96 to Cys
- CCGAATAATT AGTGCACTTT TCGAAGTCA 29(SEQ ID NO:18)
- or
- CCGAATAATT AGTACACTTT TCGAAGTCA 29(SEQ ID NO:19)
For this variant of interferon-.gamma. also the advantages illustrated in
the previous Examples resulted. In this case also this variant of
interferon-.gamma. with an antiviral activity which corresponds to about
30% of the unaltered recombinant human interferon-.gamma., is suitable for
the applications according to the invention as a therapeutic agent or as a
fine chemical such for example as for the stimulation of cell cultures.
EXAMPLE 5
A variant of the altered recombinant human interferon-.gamma. shown in
Example 1 was produced, in which the amino acids Glu8 and Ser70 were
exchanged for cystein, and in which in addition, in an otherwise known
way, the amino acid sequence of the monomeric polypeptide C-terminal was
shortened by 10 amino acids.
With increased thermal stability, this variant of the recombinant human
interferon-.gamma. reveals an activity which lies 1-4 times higher than
the antiviral activity of the unaltered recombinant human
interferon-.gamma.. Thus a variant of interferon-.gamma. is presented
which, in addition to increased thermal stability, simultaneously shows
increased biological activity.
__________________________________________________________________________
# SEQUENCE LISTING
- - - - <160> NUMBER OF SEQ ID NOS: 19
- - <210> SEQ ID NO 1
<211> LENGTH: 143
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
- - - - <400> SEQUENCE: 1
- - Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Le - #u Lys Lys Tyr Phe
Asn
1 5 - # 10 - # 15
- - Ala Gly His Ser Asp Val Ala Asp Asn Gly Th - #r Leu Phe Leu Gly Ile
20 - # 25 - # 30
- - Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Ly - #s Ile Met Gln Ser Gln
35 - # 40 - # 45
- - Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys As - #n Phe Lys Asp Asp Gln
50 - # 55 - # 60
- - Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Gl - #u Asp Met Asn Val Lys
65 - # 70 - # 75 - # 80
- - Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp As - #p Phe Glu Lys Leu Thr
85 - # 90 - # 95
- - Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Ar - #g Lys Ala Ile His Glu
100 - # 105 - # 110
- - Leu Ile Gln Val Met Ala Glu Leu Ser Pro Al - #a Ala Lys Thr Gly Lys
115 - # 120 - # 125
- - Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Ar - #g Arg Ala Ser Gln
130 - # 135 - # 140
- - - - <210> SEQ ID NO 2
<211> LENGTH: 144
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (8)
<223> OTHER INFORMATION: Xaa = Glu or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (18)
<223> OTHER INFORMATION: Xaa = Ala or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (49)
<223> OTHER INFORMATION: Xaa = Gln or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (70)
<223> OTHER INFORMATION: Xaa = Ser or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (81)
<223> OTHER INFORMATION: Xaa = Lys or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (96)
<223> OTHER INFORMATION: Xaa = Leu or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (112)
<223> OTHER INFORMATION: Xaa = His or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (121)
<223> OTHER INFORMATION: Xaa = Leu or Cys
- - <400> SEQUENCE: 2
- - Met Gln Asp Pro Tyr Val Lys Xaa Ala Glu As - #n Leu Lys Lys Tyr Phe
1 5 - # 10 - # 15
- - Asn Xaa Gly His Ser Asp Val Ala Asp Asn Gl - #y Thr Leu Phe Leu Gly
20 - # 25 - # 30
- - Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Ar - #g Lys Ile Met Gln Ser
35 - # 40 - # 45
- - Xaa Ile Val Ser Phe Tyr Phe Lys Leu Phe Ly - #s Asn Phe Lys Asp Asp
50 - # 55 - # 60
- - Gln Ser Ile Gln Lys Xaa Val Glu Thr Ile Ly - #s Glu Asp Met Asn Val
65 - # 70 - # 75 - # 80
- - Xaa Phe Phe Asn Ser Asn Lys Lys Lys Arg As - #p Asp Phe Glu Lys Xaa
85 - # 90 - # 95
- - Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gl - #n Arg Lys Ala Ile Xaa
100 - # 105 - # 110
- - Glu Leu Ile Gln Val Met Ala Glu Xaa Ser Pr - #o Ala Ala Lys Thr Gly
115 - # 120 - # 125
- - Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gl - #y Arg Arg Ala Ser Gln
130 - # 135 - # 140
- - - - <210> SEQ ID NO 3
<211> LENGTH: 143
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (8)
<223> OTHER INFORMATION: Xaa = Glu or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (18)
<223> OTHER INFORMATION: Xaa = Ala or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (49)
<223> OTHER INFORMATION: Xaa = Gln or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (81)
<223> OTHER INFORMATION: Xaa = Lys or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (96)
<223> OTHER INFORMATION: Xaa = Leu or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (70)
<223> OTHER INFORMATION: Xaa = Ser or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (112)
<223> OTHER INFORMATION: Xaa = His or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (121)
<223> OTHER INFORMATION: Xaa = Leu or Cys
<220> FEATURE:
<221> NAME/KEY: SITE
<222> LOCATION: (135)..(143)
<223> OTHER INFORMATION: Some or all of these - #residues may be missing
beginning with the c-terminal end - #of the protein.
- - <400> SEQUENCE: 3
- - Met Gln Asp Pro Tyr Val Lys Xaa Ala Glu As - #n Leu Lys Lys Tyr Phe
1 5 - # 10 - # 15
- - Asn Xaa Gly His Ser Asp Val Ala Asp Asn Gl - #y Thr Leu Phe Leu Gly
20 - # 25 - # 30
- - Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Ar - #g Lys Ile Met Gln Ser
35 - # 40 - # 45
- - Xaa Ile Val Ser Phe Tyr Phe Lys Leu Phe Ly - #s Asn Phe Lys Asp Asp
50 - # 55 - # 60
- - Gln Ser Ile Gln Lys Xaa Val Glu Thr Ile Ly - #s Glu Asp Met Asn Val
65 - # 70 - # 75 - # 80
- - Xaa Phe Phe Asn Ser Asn Lys Lys Lys Arg As - #p Asp Phe Glu Lys Xaa
85 - # 90 - # 95
- - Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gl - #n Arg Lys Ala Ile Xaa
100 - # 105 - # 110
- - Glu Leu Ile Gln Val Met Ala Glu Xaa Ser Pr - #o Ala Ala Lys Thr Gly
115 - # 120 - # 125
- - Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gl - #y Arg Arg Ala Ser
130 - # 135 - # 140
- - - - <210> SEQ ID NO 4
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 4
- - taaggttttc tgcacatttt acatatggg - # - #
29
- - - - <210> SEQ ID NO 5
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 5
- - taaggttttc tgcgcatttt acatatggg - # - #
29
- - - - <210> SEQ ID NO 6
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 6
- - tgatggtctc cacacacttt tggatgctc - # - #
29
- - - - <210> SEQ ID NO 7
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 7
- - tgatggtctc cacgcacttt tggatgctc - # - #
29
- - - - <210> SEQ ID NO 8
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 8
- - catctgaatg accgcaatta aaatatttc - # - #
29
- - - - <210> SEQ ID NO 9
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 9
- - catctgaatg accacaatta aaatatttc - # - #
29
- - - - <210> SEQ ID NO 10
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 10
- - acttggatga gttcgcatat tgctttgcg - # - #
29
- - - - <210> SEQ ID NO 11
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 11
- - acttggatga gttcacatat tgctttgcg - # - #
29
- - - - <210> SEQ ID NO 12
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 12
- - tgctattgaa aaaacagaca ttcatgtct - # - #
29
- - - - <210> SEQ ID NO 13
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 13
- - tgctattgaa aaagcagaca ttcatgtct - # - #
29
- - - - <210> SEQ ID NO 14
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 14
- - tagctgctgg cgaacattca gccatcact - # - #
29
- - - - <210> SEQ ID NO 15
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 15
- - tagctgctgg cgagcattca gccatcact - # - #
29
- - - - <210> SEQ ID NO 16
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 16
- - aaaaggagac aatgcagctc tgcattatt - # - #
29
- - - - <210> SEQ ID NO 17
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 17
- - aaaaggagac aatacagctc tgcattatt - # - #
29
- - - - <210> SEQ ID NO 18
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 18
- - ccgaataatt agtgcacttt tcgaagtca - # - #
29
- - - - <210> SEQ ID NO 19
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
- - <400> SEQUENCE: 19
- - ccgaataatt agtacacttt tcgaagtca - # - #
29
__________________________________________________________________________
Top